CN106755151B - Method for producing ARA by utilizing microbial fermentation - Google Patents

Method for producing ARA by utilizing microbial fermentation Download PDF

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CN106755151B
CN106755151B CN201710097797.5A CN201710097797A CN106755151B CN 106755151 B CN106755151 B CN 106755151B CN 201710097797 A CN201710097797 A CN 201710097797A CN 106755151 B CN106755151 B CN 106755151B
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fermentation
ara
tank
aeration
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CN106755151A (en
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文昌
陈必钦
王炳荣
王跃飞
蒋四富
徐鲁明
吴轶
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INNER MONGOLIA KINGDOMWAY PHARMACEUTICAL CO Ltd
Xiamen Kingdomway Group Co
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INNER MONGOLIA KINGDOMWAY PHARMACEUTICAL CO Ltd
Xiamen Kingdomway Group Co
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Priority to CN202010450428.1A priority Critical patent/CN111560404A/en
Priority to CN201710097797.5A priority patent/CN106755151B/en
Priority to CN202010449263.6A priority patent/CN111575323A/en
Publication of CN106755151A publication Critical patent/CN106755151A/en
Priority to PCT/CN2017/087749 priority patent/WO2018152984A1/en
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/06—Production of fats or fatty oils from raw materials by pressing
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/008—Refining fats or fatty oils by filtration, e.g. including ultra filtration, dialysis
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/10—Refining fats or fatty oils by adsorption
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/12—Refining fats or fatty oils by distillation
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/16—Refining fats or fatty oils by mechanical means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone

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Abstract

The invention belongs to the field of fermentation engineering, relates to a method for producing ARA by utilizing microbial fermentation, and particularly relates to a method for producing arachidonic acid (ARA) mixed oil by utilizing industrial fermentation of mortierella alpina strains. More specifically, the present invention relates to a method of culturing a microorganism for producing ARA, wherein: a method of culturing a microorganism for the production of ARA, wherein: starting from 100-140 hours of culture in the fermentation tank, controlling the fermentation temperature at 20-25 ℃; and/or, starting from 8-10 days of the fermenter culture, stopping aeration or reducing the aeration amount by more than 50%. The method for preparing the ARA has high yield and high purity, and is favorable for large-scale industrial production of the ARA.

Description

Method for producing ARA by utilizing microbial fermentation
Technical Field
The invention belongs to the field of fermentation engineering, relates to a method for producing ARA by utilizing microbial fermentation, and particularly relates to a method for producing arachidonic acid (ARA) mixed oil by utilizing industrial fermentation of mortierella alpina strains.
Background
ARA, also known as arachidonic acid, is a widely known name for eicosatetraenoic acid (ARA) which is a polyunsaturated fatty acid of the omega-6 series. ARA is an important essential fatty acid for human body, and is also a polyunsaturated fatty acid with highest content and most extensive distribution in human body. ARA plays an important role as a phospholipid-bound structural lipid in blood, liver, muscle and other organ systems, and ARA is a direct precursor of many circulating eicosanoic acid derivatives. These bioactive substances have important regulatory effects on lipid protein metabolism, hemorheology, vascular elasticity, leukocyte function, platelet activation, and the like. In addition, research shows that ARA has better effects of reducing blood fat, blood pressure and blood cholesterol than linoleic acid and linolenic acid; simultaneously, the medicine can relieve arrhythmia caused by barium chloride, aconitine and the like, and has better effect than linoleic acid and linolenic acid. Thus, ARA has important nutritional, health, and medical functions.
At present, ARA is mainly produced by microbial fermentation. The patent with publication number CN105112466A discloses a method for preparing arachidonic acid by fermentation with addition of a product accelerator, wherein the content of ARA in fermentation liquor is detected to be 5.41g/L after the culture of a shake flask with the addition of the product accelerator is finished, and the yield is improved by 45.1 percent compared with a control. Patent publication No. CN102925502A discloses an industrial method for producing arachidonic acid oil and fat by using Mortierella alpina, wherein the yield is 10g/L fermentation broth with unit yield of arachidonic acid. The patent with the publication number of CN104278107A discloses a method for producing arachidonic acid oil by fermenting mortierella alpina based on dissolved oxygen regulation, and the disclosed yield is 25m3The dry weight of the mortierella alpina cells, the oil content, the percentage content of the arachidonic acid in the total oil and the unit yield of the arachidonic acid in fermentation tank fermentation can respectively reach 55g/L, 54 percent and 65 percentPercent and 20.628g/L, the production intensity of the arachidonic acid reaches 1.753 g/(L.d), which is also the highest production level of the large-scale industrial production of ARA by using mortierella alpina reported at present. Although the productivity of ARA is greatly improved compared with the prior research, the production cost is greatly reduced and the unit yield is improved for the industrial production of arachidonic acid by utilizing mortierella alpina, so that the method for industrially producing ARA by microbial fermentation is far from being popularized and used widely.
The prior method for extracting ARA from the mortierella alpina fermentation culture mainly comprises three methods, namely a centrifugal method, an organic solvent extraction method and a supercritical extraction method. Centrifugation, such as patent publication No. CN1282745C, discloses a method for obtaining an oil from microbial cells, said oil comprising one or more unsaturated fatty acids, the method comprising: (a) lysing cell walls of the microbial cells to release oil; and (b) separating the oil from at least a portion of the cell wall fragments formed in (a) by centrifugation. However, the oil layer after centrifugation is poor in quality, contains water, culture medium components and cell debris besides oil and fat, and is not beneficial to subsequent refining, and in addition, the waste water layer after centrifugation contains a large amount of mushroom dregs, so that the COD is high, and the treatment is difficult or the treatment cost is extremely high. An organic solvent extraction method, such as patent with publication number CN101109015B, discloses a preparation method of peanut tetraenoic acid grease, wherein the grease extraction method comprises the following steps: and collecting, drying and grinding the aged thalli, extracting for 12 hours by using petroleum ether and ethanol, and removing the petroleum ether and the ethanol by reduced pressure distillation to obtain the arachidonic acid grease. However, in the method, an organic solvent is used for extraction, the final product may have solvent residues, and potential safety hazards such as flammability and explosiveness exist in the extraction process. The supercritical extraction method disclosed in patent publication No. CN101579019A is a method of extracting CO2The supercritical extraction method for extracting polyunsaturated fatty acid oil comprises the following steps: crushing and sieving; heating and extracting; ③ reacting liquid CO2Injecting into an extraction kettle, and pressurizing; fourthly, collecting the grease after the extraction is finished. But the supercritical equipment is expensive, the extraction yield is not high, and the oil extraction rate of the invention is only 50 percent at most.
In the prior art, the ARA crude oil is refined by adopting a chemical refining technology, and the ARA crude oil is subjected to degumming, alkali refining, decoloring and deodorizing to obtain the ARA essential oil. The process technology inevitably has some problems, such as: alkali refining in order to meet the requirement of low acid value control, excessive alkali is usually added, and partial triglyceride is inevitably saponified; high COD wastewater generated by alkali refining pollutes the environment; the alkali refining needs long high-temperature treatment time, and the peroxide value and the anisidine value of the product are easily increased; high deodorization temperature, long deodorization time, easy generation of trans-fatty acid and the like.
At present, new ARA production processes need to be developed.
Disclosure of Invention
The present inventors have made intensive studies and creative efforts to obtain a method for culturing a microorganism for producing ARA. The present inventors have surprisingly found that the cultivation method can significantly increase biomass and ARA production. Further, the present inventors have also found a method for extracting ARA crude oil, which can improve the extraction yield and quality of ARA crude oil. Further, the inventor also finds a method for purifying ARA crude oil, which can improve various technical indexes and purification yield of ARA finished oil. The invention obviously improves the yield of ARA-containing crude oil and fat, the yield of ARA and the ARA productivity. The following invention is thus provided:
one aspect of the present invention relates to a method of culturing a microorganism for producing ARA, wherein:
starting from the fermentation tank for 100-140 hours, the fermentation temperature is maintained at 20-25 deg.C (e.g., 21-25 deg.C, 20-24 deg.C, 21-23 deg.C, 22-25 deg.C, 22-24 deg.C, 23-25 deg.C, 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C or 25 deg.C); preferably, until the end of fermentation;
and/or
Stopping aeration or reducing the aeration amount by 50% or more (for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) from the 8 th to 10 th day of the fermenter culture; preferably, until the end of fermentation.
In one embodiment of the present invention, the culture method, wherein:
starting from 135 hours, 130 hours, 115 hours, 125 hours (e.g., 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 hours) or 120 hours of the fermentation tank culture, the fermentation temperature is controlled at 20-25 ℃ (e.g., 21-25 ℃, 20-24 ℃, 21-23 ℃, 22-25 ℃, 22-24 ℃, 23-25 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃ or 25 ℃), and/or
The aeration is stopped or the aeration amount is reduced by 50% or more (for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) from the 8 th, 9 th or 10 th day of the fermenter culture.
In one embodiment of the present invention, the culture method, wherein, before maintaining the fermentation temperature at 20-25 ℃, the fermentation temperature is 25-30 ℃ (e.g., 26-30 ℃, 25-29 ℃, 26-28 ℃, 27-30 ℃, 26-29 ℃, 28-30 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ or 30 ℃).
In one embodiment of the present invention, the culture method, wherein the fermentation broth has a pH of 6.0 to 7.0;
preferably, the glucose concentration in the fermentation broth is maintained at 1-5 g/L (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5g/L) before the aeration is stopped or the aeration is reduced by more than 50%.
In one embodiment of the present invention, the culture method, wherein, in stopping aeration or reducing the aeration amount by 50% or more, the fermentation broth glucose concentration is 1-2 g/L;
preferably, the fermentation temperature is 20-25 ℃ while stopping aeration or reducing the aeration amount by more than 50%;
preferably, the agitation is stopped while or after stopping the aeration or reducing the aeration amount by 50% or more;
preferably, the 3 conditions, i.e., glucose concentration, fermentation temperature, and stop stirring, are maintained until the end of fermentation;
preferably, the fermentation broth is filtered off and an appropriate amount of water or fresh fermentation medium is added before the aeration is stopped or the aeration is reduced by more than 50%.
In one embodiment of the present invention, the method for culturing, wherein the fermentor culture further comprises a step of conducting split culture 36-60 hours (e.g., 40-56 hours, 42-54 hours, 44-52 hours, 46-50 hours, 46, 47, 48, 49 or 50 hours) from the beginning of the fermentor culture; for example, the fermenter culture is carried out in two or more fermenters.
In one embodiment of the invention, the cultivation method further comprises the steps of inoculation and seed expansion culture before the fermentation tank cultivation;
preferably, the seed expansion culture comprises a primary seed expansion culture and a secondary seed expansion culture;
preferably, the primary seed expansion culture comprises the following steps:
inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4-1%, culturing at 25-32 deg.C with ventilation of 1-2 vvm and tank pressure of 0.02-0.05 MPa for 30-35 h to complete first-stage seed amplification culture;
preferably, the secondary seed expansion culture comprises the following steps:
inoculating the seed liquid of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 1-3%, culturing at 25-32 ℃, ventilating 1-2 vvm and tank pressure of 0.02-0.05 MPa for 20-25 h, and completing the second-stage seed amplification culture.
In one embodiment of the present invention, the cultivation method further comprises the step of performing activation cultivation before inoculation and seed expansion cultivation; preferably, the temperature of the activation culture is 25-32 ℃, the stirring speed is 100-.
In one embodiment of the present invention, the cultivation method, wherein the fermenter cultivation comprises the steps of:
inoculating the seed solution of the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 1-3%, culturing at 20-30 deg.C, ventilation amount of 1-2 vvm, tank pressure of 0.02-0.05 MPa, and stirring speed of 0-50 rpm, and culturing in the fermentation tank.
In one embodiment of the present invention, the culture method, wherein the microorganism used for producing ARA is mortierella alpina or a mutant strain thereof; the mortierella alpina may be one known in the art, for example, selected from strains having a collection number of CCTCC No. m2012073, CCTCC No. m2013392, CCTCC No. m2015421 and ATCC No. 42430.
In a specific embodiment of the present invention, the cultivation method comprises the following steps:
1) inoculating the slant preserved strain of Mortierella alpina (Mortierella alpina) into a 2L shake flask containing 400mL of culture medium, and culturing at 25-32 deg.C and 150rpm for 40-48 h to complete strain activation culture;
2) inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4-1%, culturing at 25-32 deg.C with ventilation of 1-2 vvm and tank pressure of 0.02-0.05 MPa for 30-35 h to complete first-stage seed amplification culture;
3) inoculating the seed solution of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 1-3%, culturing at 25-32 ℃, ventilating 1-2 vvm and tank pressure of 0.02-0.05 MPa for 20-25 h to complete the second-stage seed amplification culture;
4) inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized culture medium according to the inoculation amount of 1-3%, wherein the culture temperature is 20-30 ℃, the ventilation amount is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, and the stirring speed is 0-50 rpm, and performing fermentation culture;
5) controlling the temperature of the tank before 120h to be 25-30 ℃, and controlling the temperature of the tank after 120h and 20-25 ℃;
6) fermenting for 48h, dividing the fermentation liquid in the fermentation tank into two parts for culture, leaving half of the fermentation liquid in the main tank for continuous culture, transferring the other half of the fermentation liquid to an auxiliary tank for culture under sterile and pressure-maintaining conditions by a pressure difference method, respectively supplementing proper amount of sterilized fresh culture medium or sterile water into the main tank and the auxiliary tank, and slightly adjusting the ventilation volume, the tank pressure and the stirring speed of the main tank and the auxiliary tank after the separated culture;
7) in the fermentation process, the glucose concentration is continuously reduced along with the growth of the thalli, and a carbon source is fed-batch to maintain the sugar point (glucose concentration) in the fermentation liquor at 1-5 g/L;
8) fermenting and culturing for 8-10 days, stopping fermentation, filtering the main and auxiliary tanks through a discharge pipeline screen, adding equal volume of primary water into the obtained thalli again, slightly stirring to resuspend the thalli, stopping stirring, stopping ventilation, maintaining the tank temperature at 20-25 ℃, controlling the sugar point of the main and auxiliary tanks to be 1-2 g/L by feeding a carbon source, and standing for 1-2 days;
without being limited by theory, the step 8) can stimulate the conversion and accumulation of short-chain and low-carbon fatty acids in the Mortierella alpina thallus to ARA, so as to improve the ARA content in the thallus;
9) putting the fermentation broth in a tank, and determining that the biomass in the fermentation broth reaches 50-60 g/L, the crude oil content reaches 50-65%, the ARA accounts for 55-60% of the total oil content, the ARA yield can reach 20.1g/L, and the ARA yield can reach 1.83 g/(L.d).
In step 7) above, the sugar point (concentration of glucose) is determined using methods known to those skilled in the art, such as biosensor determination.
In one embodiment of the invention, the cultivation process is described wherein 20% to 40% (mass of crude glycerol/mass of carbon source x 100%) of the pretreated biodiesel by-product crude glycerol is added to the carbon source of the fermentation medium used. The biodiesel is renewable energy prepared by using renewable oil such as vegetable oil, animal oil and the like as raw materials, and can generate the biodiesel and a byproduct glycerol after transesterification. The pretreatment process of the crude glycerol comprises adjusting the pH value to acidity, diluting, hydrolyzing and separating. In one embodiment of the present invention, the pretreatment of crude glycerol comprises the steps of: i) mixing crude glycerol and deionized water at a volume ratio of 1: 4; ii) adjusting the pH to about 6.5 with hydrochloric acid; iii) separating and removing the precipitated substances at a rotation speed of 5000 rpm. Without being bound by theory: in step i), the viscosity can be reduced after dilution; in step ii), converting soluble saponins in the crude glycerol to insoluble free fatty acid solids; in step iii), the precipitated material comprises free fatty acid solids and insoluble heavy metal impurities.
The seed culture medium may be a seed culture medium for culturing Mortierella alpina (Mortierella alpina) in the prior art. The fermentation medium may be a fermentation medium for culturing Mortierella alpina (Mortierella alpina) in the prior art.
The carbon source in the fermentation medium comprises one or more of glucose, corn steep liquor powder, molasses, glycerol and starch; the nitrogen source comprises one or more of soybean powder, yeast powder, peptone, ammonia water, sodium nitrate, sodium glutamate and ammonium sulfate.
The microelements added into the fermentation medium comprise alanine, glutamic acid, lysine, calcium pantothenate, biotin, and vitamin B1Microorganism B6Microorganism B12And one or more of vitamin K, wherein when the vitamin K is one of the vitamin K and the vitamin K, the addition amount is 0.001% -0.01%; when the amount of the components is more than one, the addition amount of any one component is 0.001-0.005%.
The inorganic salt added into the fermentation medium comprises one or more of magnesium sulfate, potassium chloride, sodium chloride, calcium chloride, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
Another aspect of the invention relates to a fermentation culture of a microorganism obtained by the cultivation process according to any one of the invention.
Yet another aspect of the invention relates to a method for extracting ARA crude oil, comprising the steps of:
1) dehydrating a fermentation culture of a microorganism for producing ARA;
2) flexibly squeezing the product obtained in the step 1) to obtain ARA crude oil.
In one embodiment of the invention, the extraction method, wherein, in step 1), the fermentation culture is a mortierella alpina fermentation culture; preferably, the fermentation culture is a fermentation culture of the invention.
In an embodiment of the present invention, in the extraction method, in step 1), the dehydration treatment is selected from any one, two or three of:
centrifugation or plate and frame filtration, first stage flexible pressing, drying such as air drying;
preferably, the dehydration treatment sequentially comprises centrifugation and first-stage flexible pressing, or sequentially comprises plate-frame filtration and airflow drying;
preferably, the inlet air temperature of the airflow drying is 110-150 ℃, and the outlet air temperature is 30-70 ℃ until the moisture content is less than 10%.
In an embodiment of the invention, in the step 1), the first-stage flexible pressing adopts a gradual pressurization mode, the set pressure is 20-40 MPa, the pressurization time is 1-6 h, and the pressure is maintained for 1-4 h after the set pressure is reached.
In an embodiment of the invention, in the step 2), the flexible pressing adopts a gradual pressurization mode, the set pressure is 50-150 MPa, the pressurization time is 1-6 h, and the pressure is maintained for 1-4 h after the set pressure is reached.
In a specific embodiment of the present invention, the extraction method comprises the following steps:
1) material distribution: and conveying a certain amount of fermentation culture to the distribution cavity by using a distributor, returning the distributor to the initial position, and waiting for distribution in the next step.
2) Primary flexible squeezing: and (3) adopting a gradual pressurization mode, wherein the pressure range of the primary flexible pressing is 20-40 MPa, the pressurization time is about 1-6 h, the set pressure is reached, the pressure is maintained for 1-4 h until no water drops flow out basically, and after the pressure is maintained for 1-4 h, the material is descended into the heavy pressure cavity, and the material is pushed into the secondary flexible pressing position.
3) Secondary flexible squeezing: adopting a gradual pressurization mode, wherein the final pressure of squeezing is 50-150 MPa, the pressurization time is about 1-6 h, the pressure is maintained for 1-4 h until no oil drops flow out basically, the squeezed ARA crude oil is collected, the pressure is relieved, a secondary pressing cage is removed, and the mushroom residue is separated from the filter cloth.
The ARA fermentation liquor can be directly subjected to primary flexible pressing, or a centrifugal method is adopted to remove a part of water, the solid content of the fermentation liquor is increased, and then the primary flexible pressing is performed, so that the pressing time can be shortened and the production capacity can be improved.
The fermentation liquor centrifugation method can be carried out by adopting one of a horizontal spiral centrifuge, a disc centrifuge and a tubular centrifuge.
In another specific embodiment of the present invention, the extraction method comprises the following steps: filtering the fermentation culture with plate-frame filter, air-drying to obtain bacterial powder, and squeezing the bacterial powder to obtain ARA crude oil.
The plate frame filtering mode is as follows: after the fermentation liquor is fed, filtering until the filter tip of the filter press is fully opened and the feeding pressure reaches 0.6-1.0 MPa, and blowing after the filtering is finished, wherein the blowing pressure is 0.3-0.6 MPa, and the blowing time is 1-3 h.
The air inlet temperature of the airflow drying is 110-150 ℃, the air outlet temperature is 30-70 ℃, and the water content of the ARA bacterial powder is controlled within 10%.
Yet another aspect of the invention relates to a ARA crude oil, which is produced by any of the extraction methods described herein.
Yet another aspect of the invention relates to a method for purifying ARA crude oil, comprising the steps of hydrating, decolorizing and molecular distilling ARA crude oil; preferably, the ARA crude oil is the ARA crude oil of the invention.
In one embodiment of the present invention, the purification method, wherein the hydration comprises the steps of:
heating ARA crude oil to 70-85 deg.C, adding water of 75-90 deg.C according to the ratio of 1kg crude oil to 50-150 g water, stirring for 10-60 min at stirring speed of 30-90 r/min, standing for 1-6 h, and removing lower layer phospholipid to obtain the hydrated oil.
In one embodiment of the present invention, the purification method, wherein the decoloring comprises the steps of:
heating the hydration product to 90-110 ℃, controlling the vacuum degree to be less than or equal to-0.07 MPa, dehydrating in vacuum for 0.5-2 h, then cooling to 60-80 ℃, adding a decolorizing agent (such as activated carbon accounting for 1% -3% of the weight of the hydration oil and activated clay accounting for 2% -4% of the weight of the hydration oil), stirring for 0.5-1 h, stopping stirring, and filtering to remove the decolorizing agent to obtain the decolorized oil.
In one embodiment of the present invention, the purification method, wherein the molecular distillation is a three-stage molecular distillation;
preferably, the molecular distillation comprises the steps of:
the decolorized oil enters three-stage molecular distillation, the first-stage vacuum degree is controlled to be less than or equal to 100Pa, the temperature is controlled to be 150-; the obtained first heavy component enters a second-stage molecular distillation, the second-stage vacuum degree is controlled to be less than or equal to 50Pa, the temperature is controlled to be 180 DEG, and the second-stage light component is removed; and the obtained second heavy component enters a third-stage molecular distillation, the third-stage vacuum degree is controlled to be less than or equal to 5Pa, the temperature is controlled to be 200-fold, and the third-stage light component is removed to obtain a third heavy component which is ARA finished oil.
Preferably, the molecular distillation is repeated 1 or more times (e.g., 2, 3, 4, or 5 times).
In a specific embodiment of the present invention, the purification method comprises the following steps:
1) hydration: heating ARA crude oil to 70-85 deg.C, adding purified water of 75-90 deg.C according to the ratio of 50-150 g purified water to 1kg crude oil, stirring for 10-60 min at stirring speed of 30-90 r/min, standing for 1-6 h, and removing lower layer phospholipid by layering to obtain hydrated oil;
2) and (3) decoloring: transferring the hydrated oil into a decoloring pot, heating to 90-110 ℃, controlling the vacuum degree to be less than or equal to-0.07 MPa, dehydrating in vacuum for 0.5-2 h, then cooling to 60-80 ℃, adding decoloring agents (activated carbon accounting for 1-3% of the weight of the hydrated oil and activated clay accounting for 2-4% of the weight of the hydrated oil), stirring and decoloring for 0.5-1 h, stopping stirring, and filtering to remove the decoloring agents to obtain decolored oil;
3) molecular distillation: and (3) allowing the decolorized oil to enter three-stage molecular distillation, controlling the first-stage vacuum degree to be less than or equal to 100Pa and the temperature to be 150-. The number of times of molecular distillation is controlled for 1-3 times until the acid value and the odor meet the standard requirements. And (4) after molecular distillation, cooling to 20-40 ℃, adding an antioxidant, and packaging to obtain the ARA finished oil.
Yet another aspect of the invention relates to an ARA product oil, which is produced by the purification process of any one of the present invention.
Yet another aspect of the invention relates to a process for producing ARA or an ARA-containing product (e.g., ARA finished oil) using a microorganism, comprising:
the method of culturing a microorganism producing ARA according to any of the present invention,
The method for extracting ARA crude oil, and/or the method for extracting ARA crude oil
The method for purifying the ARA crude oil provided by the invention.
In one embodiment of the present invention, a process flow for producing ARA product oil is shown in fig. 1.
In another embodiment of the present invention, a process flow for producing ARA finished oil is shown in fig. 2.
In the present invention,
the term "flexible press" refers to a high-pressure press mode in which a predetermined pressure is gradually reached by performing a pressure-holding-pressure cycle using a PLC (programmable logic controller) program control.
The term "purified water" refers to water for pharmaceutical use which is obtained by subjecting drinking water to distillation, ion exchange, reverse osmosis or other suitable method, and does not contain any additives. In one embodiment of the invention, the purified water is according to the regulations of the "Chinese pharmacopoeia".
The term "ARA crude oil" refers to a primary oil obtained from ARA fermentation broth without refining.
The term "ARA finished oil" refers to essential oil obtained by refining ARA crude oil.
The term "fermentor culture" refers to a fermentation culture performed in a fermentor for the production of a product of interest following seed expansion.
In the present invention, when "aeration" or "aeration amount" is referred to, if not specifically stated, it means aeration or an amount of aeration.
Advantageous effects of the invention
The invention has at least one of the following technical effects:
(1) the technological indexes of the invention are obviously superior to those of the existing technology, the obtained ARA grease has high yield and high purity, is beneficial to large-scale industrial production of ARA, the addition of the crude glycerol also reduces the production cost of ARA fermentation, and greatly improves the market competitiveness of the ARA fermentation production.
(2) According to the invention, the ARA crude oil is prepared by adopting a flexible squeezing process, an organic solvent is not required for extraction, the whole production process route does not need to use the organic solvent, the final product does not have solvent residue, and on one hand, the obtained product is green and healthy and has good product quality, and on the other hand, a production workshop is safe and environment-friendly, so that the ARA crude oil is a green clean production process.
(3) The water solution removed by the primary flexible squeezing of the fermentation culture does not contain bacterial residues basically, has low COD and is easy for biochemical treatment, and after the secondary flexible squeezing is carried out to obtain crude oil, the residual bacterial residues also contain a small amount of grease and a large amount of protein, so that the water solution can be used as a feed additive, and is economic and environment-friendly.
(4) The invention adopts a molecular distillation one-step process to replace the traditional two-step processes of deacidification and deodorization. Molecular distillation can rapidly remove a large amount of free fatty acid and odor on the basis of keeping the physiological activity of the substance. Compared with the traditional alkali-refining deacidification method, the molecular distillation deacidification method has the advantages that the molecular distillation deacidification process is simple, the risk of excessive alkali-refining is reduced, the loss of neutral oil taken away from soapstock is reduced, the deacidification yield is obviously improved, the deacidification process is carried out under the high vacuum condition for a short time, the risk of increasing peroxide value and anisidine value caused by long heating time of alkali-refining deacidification is avoided, and the product stability is good; compared with the traditional steam distillation deodorization process, the molecular distillation deodorization time is short, the vacuum degree is high, the generation of trans-fatty acid is reduced, the deodorization effect of odor substances is good, and the product has no fishy smell.
(5) The production process avoids the use of organic solvent, avoids the cost of solvent consumption and solvent recovery, has low COD in the sewage, is easy to treat, has high refining yield, and greatly reduces the production cost.
Drawings
FIG. 1: the process flow of one embodiment of the invention for producing ARA product oil is schematic.
FIG. 2: a schematic of a process flow for producing ARA finished oil according to another embodiment of the invention.
FIG. 3: the disclosed Mortierella alpina alpine is cultured in different culture modes for 100m3Results of fermentation in a fermenter for producing ARA.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The method for measuring or calculating the physical quantity or index according to the present invention is carried out by the following method, unless otherwise specified:
method for determining biomass: taking a proper amount of fermentation culture (fermentation liquid), placing the fermentation culture (fermentation liquid) in a flat weighing bottle, drying the fermentation culture (fermentation liquid) in an electrothermal constant-temperature drying oven at 105 ℃ for 4 hours, placing the dried fermentation culture (fermentation liquid) in a dryer, cooling the dried fermentation culture (fermentation liquid) to room temperature, weighing, subtracting the weight of the weighing bottle, and dividing by the volume of the fermentation liquid to obtain a numerical value, namely biomass in unit g/L.
The method for measuring the yield of the crude oil comprises the following steps: taking a certain volume of fermentation culture (fermentation liquor), adding concentrated hydrochloric acid with the volume being 2 times of that of the fermentation culture, stirring for 50min at a constant temperature of 70 ℃ until thalli are completely digested, adding a proper amount of normal hexane, standing for layering, taking an upper layer organic phase into an eggplant-shaped bottle by using a dropper, continuously extracting for 5-8 times until the upper layer organic phase is colorless, removing the normal hexane through water bath rotary evaporation at 80 ℃, then placing the eggplant-shaped bottle into an electrothermal constant-temperature drying oven at 105 ℃ for drying for 1h, placing the eggplant-shaped bottle into a dryer for cooling to room temperature, weighing, subtracting the weight of the eggplant-shaped bottle, and dividing by the volume of the fermentation liquor to obtain a numerical value, namely the crude oil yield.
ARA yield: the ARA content in the crude oil is measured by gas chromatography and multiplied by the yield of the crude oil, and the unit is g/L.
ARA productivity: ARA production divided by the fermentation period (days) in g/(L.d).
The fatty acid component analysis method and the ARA product oil detection method are carried out according to GB 26401-2011 arachidonic acid oil (fermentation method).
The crude oil extraction yield calculation mode is as follows: the yield is g of crude oil weight/(volume L of fermentation liquor multiplied by g/L of crude oil yield of fermentation liquor multiplied by 100 percent.
3The following examples 1-5 relate to the fermentative production of ARA in a 100m fermenter.
In the following examples 1 to 5, if not otherwise specified:
the strain used was Mortierella alpina alpine (ATCC No. 42430);
the formula of the seed culture medium is as follows: potato (peeled) 200g, glucose 20g, agar 15-20 g, distilled water 1000ml, natural pH;
the formula of the fermentation medium is as follows: 22g/L glucose, 12g/L yeast powder, 8g/L peptone, 15g/L sodium chloride, 6g/L ammonium sulfate and KH2PO4 5g/L，CuSO4·5H2O 1.5μg/L，MnSO4 2μg/L，ZnSO4·7H2O 3μg/L，pH 6－7。
Example 1
The strain preserved on the slant of Mortierella alpina (Mortierella alpina) is inoculated into a 2L shake flask containing 400mL of culture medium, and cultured at a temperature of 28 ℃ and a rotation speed of 150rpm for 48h to complete strain activation culture. Inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4%, and culturing for 30h at the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the first-stage seed amplification culture. Inoculating the seed solution of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 3%, and culturing for 24h under the conditions of the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the second-stage seed amplification culture. And inoculating the seed solution in the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 3%.
The fermentation process is carried out at a culture temperature of 28 ℃, an air flow of 1vvm, a tank pressure of 0.04MPa and a stirring speed of 50rpm, and the fermentation process is carried out by feeding a carbon source containing 30% of pretreated crude glycerol and controlling the glucose concentration at about 5 g/L. And detecting the glucose concentration, pH, thallus biomass, crude oil yield and ARA yield change of the fermentation liquor in the fermentation process.
After 9 days of culture, the fermentation was terminated, and the biomass in the fermentation broth was measured to be 43g/L, the crude oil yield was measured to be 21.5g/L, the ARA yield was measured to be 10.1g/L, and the ARA productivity was measured to be 1.12 g/(L.d), as shown in FIG. 3. The volume of the fermentation liquor in the tank is 83m3The yield of ARA-containing crude oil in the whole batch of fermentation is 1784.5 kg. Table 1 shows the results of gas phase analysis of fatty acid composition of the mixed fats and oils obtained after fermentation.
Table 1: fatty acid composition of the Mixed fat obtained in example 1
Fatty acid composition Content%
C14:0 0.27
C15:0 0.12
C16:0 17.63
C17:0 0.46
C18:0 19.13
C18:1 5.34
C18:2 4.61
C18:3 4.47
C20:0 0.99
C20:4 46.98
Example 2
The strain preserved on the slant of Mortierella alpina (Mortierella alpina) is inoculated into a 2L shake flask containing 400mL of culture medium, and cultured at a temperature of 28 ℃ and a rotation speed of 150rpm for 48h to complete strain activation culture. Inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4%, and culturing for 30h at the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the first-stage seed amplification culture. Inoculating the seed solution of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 3%, and culturing for 24h under the conditions of the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the second-stage seed amplification culture. And inoculating the seed solution in the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 3%.
The aeration rate in the fermentation process is 1vvm, the tank pressure is 0.04MPa, the stirring speed is 50rpm, the tank temperature before 120h is controlled at 28 +/-1 ℃, the tank temperature after 120h and the tank temperature after 120h are controlled at 22 +/-1 ℃, a carbon source containing 30 percent of pretreated crude glycerol is fed, the glucose concentration is controlled at about 5g/L, and the fermentation culture is carried out. And detecting the glucose concentration, pH, thallus biomass, crude oil yield and ARA yield change of the fermentation liquor in the fermentation process.
After 9 days of culture, the fermentation was terminated, and the biomass in the fermentation broth was determined to be 50g/L, the crude oil yield was determined to be 29g/L, the ARA yield was determined to be 15.1g/L, and the ARA productivity was determined to be 1.68 g/(L.d), as shown in FIG. 3. The volume of the fermentation liquor in the tank is 82m3The yield of the ARA-containing crude oil in the whole batch of fermentation is 2378kg, which is 0.33 times higher than that of the example 1. The following table 2 shows the gas phase analysis results of the fatty acid composition of the mixed fat obtained after fermentation.
Table 2: fatty acid composition of the Mixed fat obtained in example 2
Fatty acid composition Content%
C14:0 0.43
C15:0 0.16
C16:0 15.21
C17:0 0.56
C18:0 16.89
C18:1 5.07
C18:2 4.51
C18:3 4.12
C20:0 0.98
C20:4 52.07
Example 3
The strain preserved on the slant of Mortierella alpina (Mortierella alpina) is inoculated into a 2L shake flask containing 400mL of culture medium, and cultured at a temperature of 28 ℃ and a rotation speed of 150rpm for 48h to complete strain activation culture. Inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4%, and culturing for 30h at the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the first-stage seed amplification culture. Inoculating the seed solution of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 3%, and culturing for 24h under the conditions of the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the second-stage seed amplification culture. And inoculating the seed solution in the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 3%.
The culture temperature in the fermentation process is 28 ℃, the ventilation volume is 1vvm, the tank pressure is 0.04MPa, the stirring speed is 50rpm, a carbon source containing 30 percent of pretreated crude glycerol is fed, the glucose concentration is controlled at 5g/L, and the fermentation culture is carried out. And (3) during fermentation for 48 hours in the fermentation process, dividing the fermentation liquor in the fermentation tank into two parts for culture, leaving one half of the fermentation liquor in the main tank for continuous culture, transferring the other half of the fermentation liquor to an auxiliary tank for culture under sterile and pressure-maintaining conditions by a pressure difference method, respectively supplementing a proper amount of sterilized fresh culture medium or sterile water into the main tank and the auxiliary tank, and slightly adjusting the ventilation volume, the tank pressure and the stirring rotation speed of the main tank and the auxiliary tank after the separated culture. And detecting the glucose concentration, pH, thallus biomass, crude oil yield and ARA yield change of the fermentation liquor in the fermentation process.
After 9 days of culture, the fermentation was terminated, and the biomass in the fermentation broth was measured to be 45g/L, the crude oil yield 22.05g/L, the ARA yield 10.6g/L, and the ARA productivity 1.18 g/(L.d), as shown in FIG. 3. The volume of the fermentation liquor in the tank is 145m3The yield of ARA-containing crude oil in the whole batch of fermentation is 3197.25kg, which is improved by 0.79 times compared with the yield in example 1, and although indexes such as tank-releasing biomass, crude oil yield, ARA yield and the like are not much different from the original culture mode, the tank-releasing volume of the fermentation liquor is 1.75 times of the original culture mode by adopting the split-tank culture, so that the yield of the whole batch of fermentation is improved by 0.79 times compared with the original culture mode. The method has important significance for industrial large-scale production of the ARA-containing grease, can greatly save the cost and improve the market competitiveness of the ARA production. The following table 3 shows the results of gas phase analysis of fatty acid composition of the mixed oils obtained after fermentation.
Table 3: fatty acid composition of Mixed fat obtained in example 3
Fatty acid composition Content%
C14:0 0.48
C15:0 0.17
C16:0 17.12
C17:0 0.52
C18:0 18.54
C18:1 5.28
C18:2 4.25
C18:3 4.36
C20:0 1.21
C20:4 48.07
Example 4
The strain preserved on the slant of Mortierella alpina (Mortierella alpina) is inoculated into a 2L shake flask containing 400mL of culture medium, and cultured at a temperature of 28 ℃ and a rotation speed of 150rpm for 48h to complete strain activation culture. Inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4%, and culturing for 30h at the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the first-stage seed amplification culture. Inoculating the seed solution of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 3%, and culturing for 24h under the conditions of the culture temperature of 28 ℃, the ventilation volume of 1vvm and the tank pressure of 0.04MPa to complete the second-stage seed amplification culture. And inoculating the seed solution in the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 3%.
The culture temperature in the fermentation process is 28 ℃, the ventilation volume is 1vvm, the tank pressure is 0.04MPa, the stirring speed is 50rpm, a carbon source containing 30 percent of pretreated crude glycerol is fed, the glucose concentration is controlled at 5g/L, and the fermentation culture is carried out. And after culturing for 9 days, stopping fermentation, filtering out fermentation liquor through a discharge pipeline screen, adding equal volume of primary water again, slightly stirring to resuspend the bacteria, stopping stirring, stopping ventilation, controlling the sugar point to be about 2g/L by feeding a carbon source, standing for 2 days, stimulating short-chain and low-carbon fatty acids in the mortierella alpina bacteria to be converted and accumulated to ARA, and improving the ARA content in the bacteria. And detecting the glucose concentration, pH, thallus biomass, crude oil yield and ARA yield change of the fermentation liquor in the fermentation process.
After 11 days of culture, the biomass in the fermentation broth was measured to be 52g/L, the crude oil and fat yield was measured to be 31.2g/L, the ARA yield was measured to be 16.5g/L, and the ARA productivity was measured to be 1.5 g/(L.d), as shown in FIG. 3. The volume of the fermentation liquor in the tank is 83m3The yield of ARA-containing crude oil in the whole batch of fermentation is 2589.6kg, which is 0.45 times higher than that of example 1. The following table 4 shows the gas phase analysis results of the fatty acid composition of the mixed oil obtained after fermentation.
Table 4: fatty acid composition of Mixed fat and oil obtained in example 4
Figure BDA0001230817250000181
Figure BDA0001230817250000191
Example 5
The strain preserved on the slant of Mortierella alpina (Mortierella alpina) is inoculated into a 2L shake flask containing 400mL of culture medium, and cultured at 25 ℃ for 24h at the rotation speed of 200rpm, thereby completing the strain activation culture. Inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4%, and culturing for 30h under the conditions of the culture temperature of 28 ℃, the ventilation volume of 1vvm, the tank pressure of 0.02MPa and the stirring speed of 50rpm to complete the first-stage seed amplification culture. Inoculating the seed solution of the first-stage seeding tank into a second-stage seeding tank filled with a sterilized culture medium according to the inoculation amount of 3%, and culturing for 24h under the conditions of the culture temperature of 28 ℃, the ventilation volume of 1vvm, the tank pressure of 0.02MPa and the stirring speed of 75rpm to complete the second-stage seed amplification culture. And inoculating the seed solution in the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 3%.
The aeration rate in the fermentation process is 1vvm, the tank pressure is 0.04MPa, the stirring speed is 50rpm, the tank temperature before 120h is controlled at 28 +/-1 ℃, the tank temperature after 120h and the tank temperature after 120h are controlled at 22 +/-1 ℃, a carbon source containing 30 percent of pretreated crude glycerol is fed, the glucose concentration is controlled at about 5g/L, and the fermentation culture is carried out. And (3) during fermentation for 48 hours in the fermentation process, dividing the fermentation liquor in the fermentation tank into two parts for culture, leaving one half of the fermentation liquor in the main tank for continuous culture, transferring the other half of the fermentation liquor to an auxiliary tank for culture under sterile and pressure-maintaining conditions by a pressure difference method, respectively supplementing a proper amount of sterilized fresh culture medium or sterile water into the main tank and the auxiliary tank, and slightly adjusting the ventilation volume, the tank pressure and the stirring rotation speed of the main tank and the auxiliary tank after the separated culture. And after culturing for 9 days, stopping fermentation, filtering out fermentation liquor by using a discharge pipeline screen in each of the main tank and the auxiliary tank, adding equal-volume primary water again, slightly stirring to resuspend the bacteria, stopping stirring, stopping ventilation, maintaining the tank temperature at 22 +/-1 ℃, controlling the sugar point of the main tank and the auxiliary tank to be about 2g/L by using a fed-batch carbon source, standing for 2 days, stimulating short-chain and low-carbon fatty acids in the mortierella alpina bacteria to be converted and accumulated to ARA, and improving the ARA content in the bacteria. And detecting the glucose concentration, pH, thallus biomass, crude oil yield and ARA yield change of the fermentation liquor in the fermentation process.
After 11 days of culture, the biomass in the fermentation broth was measured to be 54g/L, the crude oil yield was measured to be 34.02g/L, the ARA yield was measured to be 20.1g/L, and the ARA productivity was measured to be 1.82 g/(L.d), as shown in FIG. 3. The volume of the fermentation liquor discharged from the tank is 146m3The yield of ARA-containing crude oil in the whole batch of fermentation is 4966.92kg, which is 1.78 times higher than that of example 1. The following table 5 shows the gas phase analysis results of the fatty acid composition of the mixed fat obtained after fermentation.
Table 5: fatty acid composition of Mixed fat obtained in example 5
Fatty acid composition Content%
C14:0 0.45
C15:0 0.11
C16:0 14.22
C17:0 0.45
C18:0 10.46
C18:1 5.28
C18:2 4.16
C18:3 4.37
C20:0 1.42
C20:4 59.08
The following examples 6-10 and comparative example 1 relate to the extraction of ARA crude oil
Example 6
Taking 200L of ARA fermentation liquor obtained in example 5, heating and inactivating, conveying the fermentation liquor to a material distribution cavity by a material distributor, distributing and wrapping, performing primary pressing after distributing, and maintaining the pressure for 2h until no water drops flow out within 2h by adopting a gradual pressurization mode until the set pressure of 30MPa is reached. Removing the primary pressing cage, replacing the secondary pressing cage, pushing into the secondary pressing position, performing secondary pressing, adopting a gradual pressing mode, keeping the pressure for 2h until the set pressure reaches 100MPa within the set 2h, keeping the pressure for 2h until no oil drops flow out basically, collecting the pressed ARA crude oil, and obtaining 6.0kg of ARA crude oil altogether, wherein the yield of the fermentation liquid is 88.2 percent. Removing the secondary pressing cage, and separating the pressed mushroom dregs from the filter cloth.
Example 7
Taking 200L of ARA fermentation liquor obtained in example 5, heating and inactivating, conveying the fermentation liquor to a material distribution cavity by a material distributor, distributing and wrapping, performing primary pressing after distributing, and maintaining the pressure for 4h until no water drops flow out within 5 h. Removing the primary pressing cage, replacing the secondary pressing cage, pushing into a secondary pressing position, performing secondary pressing, adopting a gradual pressing mode, keeping the pressure for 4h until no oil drops flow out basically, collecting the pressed ARA crude oil to obtain 6.2kg of ARA crude oil, and obtaining the fermentation liquor with the crude oil yield of 91.1%. Removing the secondary pressing cage, and separating the pressed mushroom dregs from the filter cloth.
Example 8
Taking 300L of ARA fermentation liquor obtained in example 5, heating for inactivation, centrifuging by adopting a butterfly centrifuge, removing centrifuged light liquid to obtain 145L of concentrated fermentation liquor, conveying the concentrated fermentation liquor to a distributing cavity by using a distributing device, distributing and wrapping, performing primary pressing after distributing is finished, and maintaining the pressure for 4h until no water drops flow out basically when the set pressure of 40MPa is reached within 5h by adopting a gradual pressurization mode. Removing the primary pressing cage, replacing the secondary pressing cage, pushing into the secondary pressing position, performing secondary pressing, adopting a gradual pressing mode, keeping the pressure for 4h until no oil drops flow out basically, collecting the pressed ARA crude oil to obtain 9.4kg of ARA crude oil, and obtaining the fermentation liquor with the crude oil yield of 92.1%. Removing the secondary pressing cage, and separating the pressed mushroom dregs from the filter cloth.
Example 9
Taking 1000L of ARA fermentation liquor obtained in example 5, heating and inactivating, then, entering a plate-and-frame filter press for filtering until the feeding pressure reaches 0.7MPa, after filtering, blowing with compressed air at the blowing pressure of 0.4MPa for 2h, and after blowing, detaching the plate frame to obtain filter mud. Setting the air inlet temperature of air flow drying to 120 ℃, setting the air outlet temperature to 40 ℃, and drying the filter mud in air flow to obtain 58.1kg of ARA bacterial powder. And (3) conveying the ARA bacterial powder into a cloth cavity, wrapping the cloth, performing flexible squeezing after the cloth is finished, adopting a gradual pressurizing mode, keeping the pressure for 2 hours until the pressure reaches 100MPa within 2 hours, basically keeping the pressure for 2 hours until no oil drops flow out, collecting squeezed ARA crude oil, and obtaining 31.8kg of ARA crude oil altogether, wherein the yield of the fermentation liquid is 93.5 percent.
Example 10
Taking 1000L of ARA fermentation liquor obtained in example 5, heating and inactivating, then, entering a plate-and-frame filter press for filtering until the feeding pressure reaches 1.0MPa, blowing with compressed air after the feeding is finished, wherein the blowing pressure is 0.6MPa, and the blowing is carried out for 1h, and detaching the plate frame after the blowing is finished to obtain filter mud. Setting the air inlet temperature of airflow drying to be 140 ℃, setting the air outlet temperature to be 50 ℃, and allowing the filter mud to enter airflow drying to obtain 55.8kg of ARA bacterial powder. And (3) conveying the ARA bacterial powder into a cloth cavity, wrapping the cloth, performing flexible pressing after the cloth is finished, maintaining the pressure for 4h until no oil drops flow out basically when the pressure reaches 150MPa within 4h in a gradual pressurizing mode, collecting the pressed ARA crude oil to obtain 32.5kg of ARA crude oil, and obtaining the fermentation liquor until the yield of the crude oil is 95.5%.
Comparative example 1
Taking 1000L of ARA fermentation liquor obtained in example 5, heating and inactivating, then, entering a plate-and-frame filter press for filtering until the feeding pressure reaches 1.0MPa, blowing with compressed air after the feeding is finished, wherein the blowing pressure is 0.6MPa, and the blowing is carried out for 1h, and detaching the plate frame after the blowing is finished to obtain filter mud. Setting the air inlet temperature of airflow drying to be 140 ℃, the air outlet temperature to be 50 ℃, and allowing the filter mud to enter airflow drying to obtain 55.6kg of ARA bacterial powder. Preheating a twin-screw squeezer to 80 ℃, adding ARA bacterial powder into the twin-screw squeezer to squeeze oil, so as to obtain 18.2kg of ARA crude oil, and fermenting the liquid until the yield of the crude oil is 53.5%.
Therefore, the crude oil yield of the method for extracting the ARA crude oil is obviously higher than that of the existing method.
The following examples 11-14 and comparative example 2 relate to the purification (refining) of ARA crude oil
Example 11
10kg of the ARA crude oil obtained in examples 6, 7 and 8 are combined and refined according to the steps of hydration, decolorization and molecular distillation.
Hydration: heating 10kg of ARA crude oil to 75 ℃, adding 1kg of purified water with the temperature of 80 ℃, stirring for 30min at the stirring speed of 30 r/min, standing for 2h, and removing the lower layer to obtain 9.76kg of hydrated oil.
And (3) decoloring: heating the hydrated oil to 100 ℃, controlling the vacuum degree to be-0.075 MPa, dehydrating in vacuum for 1h, then cooling to 70 ℃, adding a decolorizing agent (191g of activated carbon and 286g of activated clay), stirring and decolorizing for 0.5h, stopping stirring, and filtering to remove the decolorizing agent to obtain 9.33kg of decolorized oil.
Molecular distillation: and (3) performing three-stage molecular distillation on the decolorized oil, controlling the first-stage vacuum degree to be about 90Pa and the temperature to be 160 ℃, removing the light components of the first stage, controlling the second-stage vacuum degree to be about 40Pa and the temperature to be 200 ℃, removing the light components of the second stage, performing third-stage molecular distillation on the heavy components, controlling the third-stage vacuum degree to be about 3Pa and the temperature to be 220 ℃, removing the light components of the third stage, collecting the heavy components, cooling to 30 ℃, adding an antioxidant, and packaging to obtain 9.13kg of ARA finished oil, wherein the inspection result is shown in Table 6.
Table 6: ARA product oil test results
Figure BDA0001230817250000231
Figure BDA0001230817250000241
Example 12
10kg of the ARA crude oil obtained in examples 6, 7 and 8 are combined and refined according to the steps of hydration, decolorization and molecular distillation.
Hydration: heating 10kg of ARA crude oil to 85 ℃, adding 1kg of purified water with the temperature of 90 ℃, stirring for 15min at the stirring speed of 90 r/min, standing for 4h, and removing the lower layer to obtain 9.81kg of hydrated oil.
And (3) decoloring: heating the hydrated oil to 110 ℃, controlling the vacuum degree to be-0.075 MPa, carrying out vacuum dehydration for 0.5h, then cooling to 80 ℃, adding a decoloring agent (192g of activated carbon and 288g of activated clay), stirring and decoloring for 1h, stopping stirring, and filtering to remove the decoloring agent to obtain 9.39kg of decolored oil.
Molecular distillation: and (3) performing three-stage molecular distillation on the decolorized oil, controlling the first-stage vacuum degree to be about 90Pa and the temperature to be 200 ℃, removing the light components of the first stage, controlling the heavy components to be about 3Pa and the temperature to be 250 ℃, removing the light components of the third stage, collecting the heavy components, cooling to 30 ℃ after the molecular distillation is finished, adding an antioxidant, and packaging to obtain 9.08kg of ARA finished oil, wherein the inspection result is shown in Table 7.
Table 7: ARA product oil test results
Figure BDA0001230817250000251
Example 13
10kg of the crude ARA oils obtained in examples 9 and 10 were combined and refined by the refining method of example 11 to obtain 9.20kg of ARA finished oil, and the results of the test are shown in Table 8.
Table 8: ARA product oil test results
Figure BDA0001230817250000252
Figure BDA0001230817250000261
Example 14
10kg of the crude ARA oils obtained in examples 9 and 10 were combined and refined by the refining method of example 12 to obtain 9.13kg of ARA finished oil, and the results of the test are shown in Table 9.
Table 9: ARA product oil test results
Figure BDA0001230817250000262
Figure BDA0001230817250000271
Comparative example 2
10kg of the ARA crude oil obtained in examples 9 and 10 were combined and refined according to the conventional refining method, i.e. hydration, alkali refining, decolorization and deodorization.
Hydration: heating 10kg of ARA crude oil to 85 ℃, adding 1kg of purified water with the temperature of 90 ℃, stirring for 15min at the stirring speed of 90 r/min, standing for 4h, and removing the lower layer to obtain 9.80kg of hydrated oil.
Alkali refining: keeping the temperature of the hydrated oil at 75 ℃, adding 1L of NaOH solution with the concentration of 10 percent (mass fraction), stirring for 30min, stirring at the speed of 30 r/min, standing for 4h, separating soapstock to obtain alkali refined oil, spraying purified water with the weight of 10 percent of the oil at 80 ℃ under the stirring condition of the alkali refined oil for washing, controlling the water adding time to be 10-30 min, standing for 2h after the water is added, separating a water layer, and repeatedly washing for 2 times to obtain 9.10kg of the alkali refined oil.
And (3) decoloring: heating the alkali refined oil to 110 ℃, controlling the vacuum degree to be-0.075 MPa, dehydrating in vacuum for 0.5h, then cooling to 80 ℃, adding a decolorizing agent (182g of activated carbon and 273g of activated clay), stirring and decolorizing for 1h, stopping stirring, and filtering to remove the decolorizing agent to obtain 8.65kg of decolorized oil.
Deodorizing: transferring the decolorized oil into a deodorizing pot, introducing steam for deodorization, controlling the deodorizing temperature at 185 ℃, controlling the vacuum degree within 600Pa, deodorizing for 2h, stopping introducing steam after deodorization, cooling to 20-40 ℃, adding an antioxidant, and packaging to obtain 8.41kg of ARA product oil, wherein the detection results are shown in Table 10.
Table 10: ARA product oil test results
Figure BDA0001230817250000281
The result shows that the method for purifying the ARA crude oil can better remove peroxide and unsaponifiable matters, avoid the generation of trans-fatty acid and has lower acid value.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (22)

1. A method of culturing a microorganism for the production of ARA, wherein:
starting from 110-130 hours of culture in a fermentation tank, wherein the fermentation temperature is 20-24 ℃; wherein the fermentation temperature is 26-30 ℃ before the fermentation temperature is 20-24 ℃;
and/or
Stopping aeration or reducing the aeration amount by more than 90% from 8-10 days of the fermentation tank culture; wherein the concentration of glucose in the fermentation broth is 1-2 g/L while or after stopping aeration or reducing the aeration amount by 90% or more; stopping stirring while or after stopping aeration or reducing the aeration amount by 90% or more; filtering out the fermentation liquid before stopping aeration or reducing the aeration amount by more than 90 percent, and adding proper amount of water or fresh fermentation medium;
wherein the microorganism for producing ARA is Mortierella alpina which is a strain deposited under ATCC No. 42430.
2. The culture method according to claim 1, wherein the fermentation temperature is maintained at 20-24 ℃ until the end of the fermentation.
3. The culture method according to claim 1, wherein aeration is kept stopped or the aeration amount is reduced by 90% or more until the end of fermentation.
4. The culture method according to claim 1, wherein:
starting from 115-125 hours of fermentation in the fermentation tank, the fermentation temperature is 20-24 ℃.
5. The culture method according to claim 1, wherein:
the fermentation temperature is 20-24 ℃ from 120 hours of the fermenter culture.
6. The culture method according to any one of claims 1 to 5, wherein:
starting from day 8, day 9 or day 10 of the fermenter culture, aeration was stopped or the aeration amount was reduced by 90% or more.
7. The culture method according to any one of claims 1 to 5, wherein the pH of the fermentation broth is 6.0 to 7.0.
8. The culture method according to any one of claims 1 to 5, wherein the concentration of glucose in the fermentation broth before stopping aeration or reducing the aeration amount by 90% or more is 1 to 5 g/L.
9. The culture method according to any one of claims 1 to 5, wherein the fermentation temperature is 20 to 24 ℃ at the same time as or after the aeration is stopped or the aeration amount is reduced by 90% or more.
10. The culture method according to any one of claims 1 to 5, wherein the fermenter culture further comprises a step of performing a split-tank culture 36 to 60 hours after the start of the fermenter culture.
11. The culture method according to claim 10, wherein the split-tank culture is a fermentor culture divided into two or more fermentors.
12. The culture method according to any one of claims 1 to 5, wherein the fermenter culture further comprises a step of performing a split tank culture at 48 hours from the start of the fermenter culture.
13. The culture method according to claim 12, wherein the split-tank culture is a fermentor culture performed in two or more fermentors.
14. The method according to any one of claims 1 to 5, further comprising the steps of inoculation and seed expansion before the fermenter culture.
15. The method according to claim 14, wherein the seed expansion culture comprises a primary seed expansion culture and a secondary seed expansion culture.
16. The method of claim 15, wherein the primary seed expansion culture comprises the steps of:
inoculating the shake flask seed solution into a first-stage seed tank filled with sterilized culture medium according to the inoculation amount of 0.4-1%, culturing at 25-32 deg.C with ventilation amount of 1-2 vvm and tank pressure of 0.02-0.05 MPa for 30-35 h to complete first-stage seed amplification culture.
17. The method of claim 15, wherein the secondary seed expansion culture comprises the steps of:
inoculating the seed liquid of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 1-3%, culturing at 25-32 ℃, ventilating 1-2 vvm and tank pressure of 0.02-0.05 MPa for 20-25 h, and completing the second-stage seed amplification culture.
18. The method according to claim 14, further comprising the step of performing activation culture before the inoculation and the seed expansion culture.
19. The culture method according to claim 18, wherein the temperature of the activation culture is 25-32 ℃, the stirring speed is 100-200 rpm, and the time is 40-48 h.
20. The cultivation method according to claim 17 or 18, wherein the fermenter cultivation comprises the following steps:
inoculating the seed solution of the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 1-3%, culturing at 20-30 deg.C, ventilation amount of 1-2 vvm, tank pressure of 0.02-0.05 MPa, and stirring speed of 0-50 rpm, and culturing in the fermentation tank.
21. A method for producing ARA or an ARA-containing product using a microorganism, comprising:
a method according to any one of claims 1 to 20 for culturing a microorganism for the production of ARA.
22. The production process according to claim 21, wherein the ARA-containing product is an ARA finished oil.
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